Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Low rank subspace clustering (LRSC)

We consider the problem of fitting a union of subspaces to a collection of data points drawn from one or more subspaces and corrupted by noise and/or gross errors. We pose this problem as a non-convex optimization problem, where the goal is to decompose the corrupted data matrix as the sum of a clean and self-expressive dictionary plus a matrix of noise and/or gross errors. By self-expressive we mean a dictionary whose atoms can be expressed as linear combinations of themselves with low-rank coefficients. In the case of noisy data, our key contribution is to show that this non-convex matrix decomposition problem can be solved in closed form from the SVD of the noisy data matrix. The solution involves a novel polynomial thresholding operator on the singular values of the data matrix, which requires minimal shrinkage. For one subspace, a particular case of our framework leads to classical PCA, which requires no shrinkage. For multiple subspaces, the low-rank coefficients obtained by our framework can be used to construct a data affinity matrix from which the clustering of the data according to the subspaces can be obtained by spectral clustering. In the case of data corrupted by gross errors, we solve the problem using an alternating minimization approach, which combines our polynomial thresholding operator with the more traditional shrinkage-thresholding operator. Experiments on motion segmentation and face clustering show that our framework performs on par with state-of-the-art techniques at a reduced computational cost.

Systemic levels of the anti-inflammatory decoy receptor soluble RAGE (receptor for advanced glycation end products) are decreased in dogs with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a common condition in dogs, and a dysregulated innate immunity is believed to play a major role in its pathogenesis. S100A12 is an endogenous damage-associated molecular pattern molecule, which is involved in phagocyte activation and is increased in serum/fecal samples from dogs with IBD. S100A12 binds to the receptor of advanced glycation end products (RAGE), a pattern-recognition receptor, and results of studies in human patients with IBD and other conditions suggest a role of RAGE in chronic inflammation. Soluble RAGE (sRAGE), a decoy receptor for inflammatory proteins (e.g., S100A12) that appears to function as an anti-inflammatory molecule, was shown to be decreased in human IBD patients. This study aimed to evaluate serum sRAGE and serum/fecal S100A12 concentrations in dogs with IBD. Serum and fecal samples were collected from 20 dogs with IBD before and after initiation of medical treatment and from 15 healthy control dogs. Serum sRAGE and serum and fecal S100A12 concentrations were measured by ELISA, and were compared between dogs with IBD and healthy controls, and between dogs with a positive outcome (i.e., clinical remission, n=13) and those that were euthanized (n=6). The relationship of serum sRAGE concentrations with clinical disease activity (using the CIBDAI scoring system), serum and fecal S100A12 concentrations, and histologic disease severity (using a 4-point semi-quantitative grading system) was tested. Serum sRAGE concentrations were significantly lower in dogs with IBD than in healthy controls (p=0.0003), but were not correlated with the severity of histologic lesions (p=0.4241), the CIBDAI score before (p=0.0967) or after treatment (p=0.1067), the serum S100A12 concentration before (p=0.9214) and after treatment (p=0.4411), or with the individual outcome (p=0.4066). Clinical remission and the change in serum sRAGE concentration after treatment were not significantly associated (p=0.5727); however, serum sRAGE concentrations increased only in IBD dogs with complete clinical remission. Also, dogs that were euthanized had significantly higher fecal S100A12 concentrations than dogs that were alive at the end of the study (p=0.0124). This study showed that serum sRAGE concentrations are decreased in dogs diagnosed with IBD compared to healthy dogs, suggesting that sRAGE/RAGE may be involved in the pathogenesis of canine IBD. Lack of correlation between sRAGE and S100A12 concentrations is consistent with sRAGE functioning as a non-specific decoy receptor. Further studies need to evaluate the gastrointestinal mucosal expression of RAGE in healthy and diseased dogs, and also the formation of S100A12-RAGE complexes.

Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells.

Virus-associated pulmonary exacerbations, often associated with rhinoviruses (RVs), contribute to cystic fibrosis (CF) morbidity. Currently, there are only a few therapeutic options to treat virus-induced CF pulmonary exacerbations. The macrolide antibiotic azithromycin has antiviral properties in human bronchial epithelial cells. We investigated the potential of azithromycin to induce antiviral mechanisms in CF bronchial epithelial cells. Primary bronchial epithelial cells from CF and control children were infected with RV after azithromycin pre-treatment. Viral RNA, interferon (IFN), IFN-stimulated gene and pattern recognition receptor expression were measured by real-time quantitative PCR. Live virus shedding was assessed by assaying the 50% tissue culture infective dose. Pro-inflammatory cytokine and IFN-β production were evaluated by ELISA. Cell death was investigated by flow cytometry. RV replication was increased in CF compared with control cells. Azithromycin reduced RV replication seven-fold in CF cells without inducing cell death. Furthermore, azithromycin increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels. While stimulating antiviral responses, azithromycin did not prevent virus-induced pro-inflammatory responses. Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway. Clinical studies are needed to elucidate the potential of azithromycin in the management and prevention of RV-induced CF pulmonary exacerbations.

Prolonged activity of the pestiviral RNase Erns as an interferon antagonist after uptake by clathrin-mediated endocytosis

The RNase activity of the envelope glycoprotein E(rns) of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E(rns) is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E(rns). Here, we show that the activity of soluble E(rns) as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E(rns) protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E(rns) mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E(rns) remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E(rns) acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase E(rns) was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E(rns) at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E(rns) is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E(rns) remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Optimized aperture shapes for depth estimation

The finite depth of field of a real camera can be used to estimate the depth structure of a scene. The distance of an object from the plane in focus determines the defocus blur size. The shape of the blur depends on the shape of the aperture. The blur shape can be designed by masking the main lens aperture. In fact, aperture shapes different from the standard circular aperture give improved accuracy of depth estimation from defocus blur. We introduce an intuitive criterion to design aperture patterns for depth from defocus. The criterion is independent of a specific depth estimation algorithm. We formulate our design criterion by imposing constraints directly in the data domain and optimize the amount of depth information carried by blurred images. Our criterion is a quadratic function of the aperture transmission values. As such, it can be numerically evaluated to estimate optimized aperture patterns quickly. The proposed mask optimization procedure is applicable to different depth estimation scenarios. We use it for depth estimation from two images with different focus settings, for depth estimation from two images with different aperture shapes as well as for depth estimation from a single coded aperture image. In this work we show masks obtained with this new evaluation criterion and test their depth discrimination capability using a state-of-the-art depth estimation algorithm.

Interferon response of the cystic fibrosis bronchial epithelium to major and minor group rhinovirus infection

Rhinoviruses (RVs) are associated with exacerbations of cystic fibrosis (CF), asthma and COPD. There is growing evidence suggesting the involvement of the interferon (IFN) pathway in RV-associated morbidity in asthma and COPD. The mechanisms of RV-triggered exacerbations in CF are poorly understood. In a pilot study, we assessed the antiviral response of CF and healthy bronchial epithelial cells (BECs) to RV infection, we measured the levels of IFNs, pattern recognition receptors (PRRs) and IFN-stimulated genes (ISGs) upon infection with major and minor group RVs and poly(IC) stimulation. Major group RV infection of CF BECs resulted in a trend towards a diminished IFN response at the level of IFNs, PRRs and ISGs in comparison to healthy BECs. Contrary to major group RV, the IFN pathway induction upon minor group RV infection was significantly increased at the level of IFNs and PRRs in CF BECs compared to healthy BECs.